Bacterial infections of the gastrointestinal (Gl) tract cause 300,000 hospitalizations and 5,000 deaths each year in the United States. We previously demonstrated that the galanin-1 receptor (Gal1R) is up-regulated in colonic epithelial cells in response to infection with diverse bacterial pathogens. When present, Gal1R activation by ligand normally present in enteric nerves results in CI- secretion, a mechanism that is responsible for a large component of the excess ntestinal fluid secretion observed in infectious diarrhea. We herein show that the increase in bacterial pathogen-induced colonic fluid secretion mediated by the GaUR occurs via a cAMP-independent but Ca2+-dependent pathway that is inhibitable by pertussis toxin, suggesting that this receptor couples to a member of the Galphi(i) family of heterotrimeric G proteins. We also provide evidence indicating that the Gal1R increases colonic CI- secretion via a calcium-activated CI- channel (CLCA). Thus we extend our novel and mechanistic hypothesis that Gal1-R up-regulation accounts for a significant component of the excessive fluid secretion observed in infectious diarrhea and propose that this occurs via a Ga\ph\(\)/CLCA-dependent pathway. To evaluate this hypothesis we will: 1. Determine how the Gal1R activates its cognate G protein(s) to cause Cl- secretion. The specific G protein(s) coupling to the Gal1R along with the receptor sites involved in this coupling will be identified. We will use the nonhydrolyzable GTP photoaffinity analog azidoanilido GTP to identify the G protein(s) physically coupling to the Gal1R. We will use the novel method of synthetic peptides on membrane support (SPOT) to obtain initial information as to where the G protein subunit(s) bind to the Gal1R. These data will be modeled using GRASP, AutoDock and DOCK to obtain initial information as to sites of Gal1R- Galphi(i) binding. These sites will be tested by introducing mini-peptides into permeabilized cells that are predicted to block Gal1R- Galphi(i) binding;those abrogating galanin-induced signaling will confirm the regions so identified as critical to this interaction. We will then: 2. Identify the Gall R-activated CI- channel mediating galanin-induced CI- secretion. Although our preliminary data strongly suggests that galanin increases colonic fluid secretion in a CFTR-independent manner by activating a CLCA chloride channel, increased amounts of colonic fluid can also result from activation of CLC chloride channels, K+ channels, or decreased Na+ absorption. To do this we will study enteric pathogen infection in wild type mice, and in mice genetically incapable of expressing Gal1R (Gal1R-/-) and in mice genetically incapable of expressing CFTR (CFTR-/-). Studying the Gal1R-/- mice will allow us to determine the contribution of galanin-mediated colonic fluid secretion, and studying the CFTR-/- will allow us to determine the direct contributions of CFTR-independent pathways. Overall these experiments will allow us to elucidate the mechanism whereby galanin causes CI- secretion, and generate information useful for the generation of agents useful in the treatment of diarrhea due to enteric pathogen infection.